Species C human adenovirus such as adenovirus type (Ad5) are used widely in therapeutic applications, for delivery of exogenous genes or as oncolytic agents. Nevertheless, many aspects of the molecular mechanisms by viral gene products affect host cell processes to allow efficient viral replication remain poorly or incompletely understood. The overall objective of the proposed studies is to improve our understanding of these mechanisms in normal and transformed human cells, in particular of the molecular functions of the E1B 55kDa protein. This protein fulfills multiple functions, yet its coding sequence is deleted from the great majority of vectors currently in use. It can repress p53-dependent transcription and in infected cells serves as the substrate recognition subunit of an infected cell-specific E3 ubiquitin-ligase (the Ad E3 ligase) that also contains the viral E4 Orf6 and several cellular proteins. The activity of this ligase is required to target p53 for proteasomal degradation, to protect infected cells against activation of the cellular doublestranded DNA break repair response and for induction of selective export of viral late mRNAs. The degree to which export of viral late mRNAs depends on the E1B 55kDa protein appears to contribute to selective replication of E1B-null mutants in tumor cells. However, the relevant substrates of the Ad E3 ligase are not known. One specific aim of the proposed studies is to identify these substrates. Export of viral late mRNAs is via the major mammalian mRNA export receptor, Nxf1 (Fig. 1). A new combination of techniques will be applied to develop an unbiased, proteomic approach to identification of novel substrates of the Ad E3 ligase. The participation of such substrates in export of viral late mRNAs will then be assessed using RNA interference to block production of the cellular proteins of interest. A recently described function of the E1B 55 kDa proteins is repression of transcription of cellular genes that encode proteins of the innate and adaptive immune responses. This function is of particular interest in the context of the immunogenicity of adenoviral vectors. Mutations that impair this function will be identified, and exploited to test the hypothesis that this function of the E1B protein modulates immune responses to adenovirus infection. The sensitivity to exogenous interferons of replication of Ad5 and E1B mutant viruses in normal human cells will be compared, as will the production of this and other cytokines following infection. The initial innate and subsequent adaptive immune responses induced when these viruses are injected into mice will also be examined. To investigate the mechanism of transcriptional repression by the E1B 55 kDa protein, such methods as chromatin immunoprecipitation, high throughput sequencing and transient expression assays will be used to determine whether the E1B 55 kDa protein associates specifically with the genes it represses or alters post-translational modification of histones that package these genes.